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Abstract

In vivo fluorescent cellular imaging of deep internal organs is highly challenging, because the excitation needs to penetrate through strong scattering tissue and the emission signal is degraded significantly by photon diffusion induced by tissue-scattering. We report that by combining two-photon Bessel light-sheet microscopy with nonlinear structured illumination microscopy (SIM), live samples up to 600 microns wide can be imaged by light-sheet microscopy with 500 microns penetration depth, and diffused background in deep tissue light-sheet imaging can be reduced to obtain clear images at cellular resolution in depth beyond 200 microns. We demonstrate in vivo two-color imaging of pronephric glomeruli and vasculature of zebrafish kidney, whose cellular structures located at the center of the fish body are revealed in high clarity by two-color two-photon Bessel light-sheet SIM.

Figures (10)

Schematic of the Bessel two-photon light-sheet microscope. (a) Optical setup. The beam path illustrated in the figure is for the case of a uniform phase pattern on the SLM. (b) Two-photon emission from a cuvette of fluorescein solution under a focused Gaussian beam, and (c) under a Bessel beam.

3D two-photon imaging of the entire vasculature of a transgenic Tg(kdrl:GFP) zebrafish head at 3 days post fertilization (dpf). The fish vasculature is labeled with GFP. (a-e) Selected x-y plane slices of two-photon Bessel light-sheet images. (f) x-z plane cross section of the 3D image stacks (g) y-z plane cross section of the 3D image stack. The full image set (512 × 512 × 577) can be viewed in Media 1. 3D images were acquired by moving the sample tube at a Nyquist sampling density of 1-μm per z step, with the imaging plane moving deeper into the fish as z increases (tube moves toward camera). The tube lens focal length was set to 70 mm, resulting in a lateral pixel size of 1.1 μm. The expose time was 1s per step. Scale bar: 100 μm.

Cellular 3D imaging by two-photon Bessel light-sheet microscope. (a) 3D rendered image of epithelial cells forming vasculature in a Tg(kdrl:GFP) zebrafish tail. The 3D image is color-coded in z-axis. (b) Cross section in the y-z plane and (c) in the x-z plane, showing vessel structures. Full 3D image (512 × 512 × 88) can be viewed in Media 2. 3D images were acquired in 1-μm z-steps by moving the sample tube, with the imaging plane moving deeper into the fish as z increases. The tube lens focal length was set to 300 mm, resulting in a lateral pixel size of 0.27 μm. The exposure time was 1s per step.

Video rate two-photon images of heartbeat and blood cell (green arrows) circulation in live Tg(kdrl:GFP) zebrafish at 2-3dpf. The video was taken with the tube lens at f = 70mm and a lateral pixel size of 1.1 μm. Areas outside of the heart were chopped in the figure. Full size video (512 × 512) is provided as Media 3. Scale bar: 50 μm.

Diffusion reduction with a modified SIM reconstruction algorithm. (a) Merged image of 5 SIM raw frames with phase-shifted patterns. The merged image is equivalent to a 10-seconds-exposure image under uniform light-sheet. (b) Raw image frame under structured light-sheet illumination; the expose time was 2s; (c) Reconstructed image by the standard SIM algorithm, showing stripe-shaped artifacts due to residual diffusion background; (d) Reconstructed image from ± 1 order harmonics by the improved SIM algorithm. Stripe-shape artifacts were removed; (e) Reconstructed image from ± 2 order harmonics by the improved SIM algorithm; (f) Weighted merge of ± 1 and ± 2 order reconstructed image. All images were taken from the kidney of a live transgenic Tg(pod:NTR-mCherry) zebrafish at 4 dpf. Podocytes in the fish kidney is labeled with mCherry. Images was taken with the tube lens at f = 135mm and a lateral pixel size of 0.6 μm. The scale bar is 10 μm.